User:Christopher French

CASPASE 8
CRYSTAL STRUCTURE OF THE COMPLEX OF CASPASE-8 WITH THE TETRAPEPTIDE INHIBITOR ACE-IETD-ALDEHYDE

Caspase 8 is a member of the caspase family, a family of cysteine proteases that play an important role in inflammation and apoptosis (programmed cell death). The caspases are essential for apoptosis in cells during development and during later stages of life. Failure of apoptosis can lead to tumor formation and the development of autoimmune diseases. In addition, excess apoptosis has been implicated in various disease states, including ischemia and Alzheimer’s. Caspase 8 is just one of 11 caspases that have been indentified in humans. Caspase 8 is an “initiator caspase” which cleave inactive pro-forms of the effector caspases which activates them. Caspases exist as inactive proenzymes that are composed of a prodomain, and a large and small protease subunit. The activation of caspase requires proteolysis at an internal aspartic residue which results in the generation of a heterodimeric enzyme with a large and small subunit.

Caspase 8 is induced by tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL). TRAIL induces apoptosis via death receptors (DR4 and DR5). After ligand binding the death receptor Fas recruits the adaptor protein FADD. FADD then binds and activates procaspase-8. Upon activation, Caspase 8 is then able activate caspase 3 and other downstream effectors. The end result is apoptosis (1,2).

Structure & Function
Caspase 8 is a 58 kilodalton protein that shares similarities with other members of the caspase family. The protein is composed of two subunits, referred to as p18 and p11. These two subunits form a heterodimer. The protein has a α/ß folding motif that has a central six stranded beta sheet. Five of the strands are parallel and one is anti-parallel. The antiparallel strand is on the edge of the ß sheet. There are also six alpha helices in the protein structure. Three of these alpha helices are located on one side of the ß sheet and the other two on the other side, forming a three layer sandwich. The p18 subunit has a Rossmann fold. There is a turn of helix (α1’) which is part of a large loop (loop 1). This is along the binding pocket region of the p18 subunit. There is a two-stranded antiparallel ß sheet found at the top of the main ß sheet which forms the base of the binding pocket (3,4).

Heterotetramer

Two p18-p11 heterodimers form a tetramer. This extends the six strands of the ß sheet to 12 strands. In the heterotetramer, the first segment of the P11 subunit and the last segment of the p18 subunit extend from the structure in an anti-parallel fashion and interact with the other heterodimer.  Residues 363-374 and 385-396 of one heterodimer extend into the other heterodimer and interact with residues Thr390 and Asp395(3).

The active site

The catalytic triad in caspase 8 comprises Cys360, His317, and Arg258 . See S1-S4 pockets. . The carboxyl group of P1 aspartate forms a salt bridge with Arg413 and Arg260. It also forms hydrogen bonds to Gln358. The P1 α-carbonyl group rotates and the oxygen atom rehybridizes to become a hydroxyl group. This then forms a hydrogen bond with the imidazole group of His317. A clear interaction exists between the carbonyl oxygen of Arg258 and the Nє of His317. The S2 pocket Cγ atom of the threonine sidechain of P2 lies in a hydrophobic pocket that is formed from the sidechains of Val410 and Tyr412. The Oγ is surrounded by water molecules. The S3 pocket consists of a glutamate at P3 that sits in a cleft made by Arg413, Arg258, and Pro415, and Asn261. Arg413 forms a salt bridge to P3 glutamate and P1 aspartate sidechains. Arg413 also forms hydrogen bonds between its main chain atoms and the mainchain if the peptide inhibitor. The S4 pocket is especially important in selectivity. The acetyl of the inhibitor hydrogen bonds to the carboxyl group of P3 glutamate through a hydrogen bond. The hydrogen bond participants then move away to accommodate a non-polar residue. The faces of two aromatic residues, Trp420 and Tyr412 help form part of the hydrophobic S4 pocket (3).

Conservation
All caspases employ a conserved cysteine residue that is used as a nucleophile for attack of peptide bonds. The sites of cleavage on the substrate all contain an aspartate at P1. The caspase catalytic domain has a mass around 30 kDa and comprises two polypeptide chains, a 17-20 kDa N-terminal fragment (α subunit) and a 10-12 kDa C-terminal fragment (ß subunit). The α subunit contains the active site cysteine and ß subunit contributes to the formation of the active site. The catalytic regions of caspase-3 and caspase-8 are similar in regards to length, placement of the active site cysteine, and pattern of processing required for activation. The main distinction is in the N-terminal prodomain of caspase-8 which contains two death-effector domains. The overall topology is similar to caspase-1 and caspase-3, with the p18 and p11 subunits folded into a compact cylinder. There are many differences among the three in the loop regions near the active site. Compared to caspase 3, caspase 8 had an insertion of seven residues designated as loop1 that lies between strand ß1 and helix α1. In caspase-1 loop 3 is longer than in caspase 8 and 3. Loop four is identical in all three caspases and loop 5 is intermediate in length between caspase 3 and 8. All three caspases adopt different conformations in this region and this has an important effect of specificity of the S4 pocket. Pockets S1-S3 are similar in all three caspases, however, the S4 pocket is different. Caspase 3 prefers an aspartate residue in the P4 position, while caspase 1 and 8 prefer hydrophobic groups. The acetyl of the inhibitor hydrogen bonds to the carboxyl group of P3 glutamate through a hydrogen bond. The S4 pocket in caspase 8 differs from caspase 3 because the hydrogen bond participants move away to accommodate a non-polar residue (3,4).



Disease
Caspase-8 may be involved in various diseases including Huntington’s disease (HD), type two diabetes, autoimmune lymphoproliferative disease, arthritis, and cancer. HD is associated with increased HD gene product called huntingtin. Overexpression of this gene product induces apoptosis in cerebellar and striatal neurons. Patients with HD have increased activated caspase 8 in the affected regions of their brains. The mutated huntingtin does not bind huntingtin interacting protein-1 (Hip-1). Free Hip-1 which is in excess then binds and initiates the apoptotic cascade through capsase-8. Thus, modulation of the caspase-8 apoptotic cascade is an treatment strategy to patients with HD. Defects is caspase-8 may also be responsible for autoimmune lymphoproliferative syndrome (ALPS). Some ALPS patients have a mutation in their caspase 8 gene that reduces protein stability and diminishes the enzymatic activity of the caspase 8 protein. Thus, caspase-8 plays a very important role in the immune system. Caspase 8 may play a role in arthritic diseases such as rheumatoid arthritis (RA). Deregulation of apoptosis in osteoblasts and T cells in rheumatoid synovium is a hallmark of RA. RA macrophages have reduced Fas-induced apoptosis. Caspase 8 may also play a role in cancer, where it acts as a tumor suppressor. Caspase-8 has been found to be downregulated in pediatric tumors and neuroendocrine lung tumors. Mutations in the caspase 8 gene have also been reported in small cell lung carcinoma. Downregulation of caspase 8 may lead to increased resistance to chemotherapy and increased metastasis. Thus, capase-8 upregulation and its inhibition are both attractive strategies for treatment in various diseases processes (5).

Links to other structures

1f9e

2k7z

1i4e

2c2z

2fun